During the investigation of the mechanism of actions of insulin-like growth factor binding protein-3 (IGFBP-3), we identified tissue transglutaminase (TG2) as the kinase responsible for the phosphorylation of IGFBP-3. IGFBP-3 is a multifunctional protein that not only functions to transport the insulin-like growth factors (IGF-I and IGF-II ) and modulate the actions of these growth factors but also has IGF-independent and anti-proliferative and proapoptotic effects (Jones and Clemmons, 1995, Endoc. Rev., 16:3–34). It can both enhance and inhibit the effects of IGF-I in vivo and in vitro depending upon experimental conditions (DeMellow and Baxter, 1988, Biochem. Biophys. Res Commun., 156:199–204; Valentinis et al., 1996, Mole. Endocrinol., 9:361–367; Oh et al., 1995, Prog. Growth Factor Res., 6:205–212; Lalou et al., 1996, Endocrinology 137:3206–3212; Hong et al., 2002, J. Biol. Chem., 277:10489–10497).
In addition to these IGF-dependent effects, emerging evidence suggests that IGFBP-3 also functions directly to stimulate apoptosis (programmed cell death) and inhibits cellular proliferation of various cell lines including human breast cancer cells (Oh et al., 1995). However these IGF-independent effects are only apparent under conditions where the IGF-I dependent effects are not observed. For example, studies with mutant IGFBP-3 and IGFBP-3 fragments which have minimal affinity for IGF-I (Lalou et al., 1996; Hong et al., 2002) and with cell lines devoid of IGF-I receptors (Valentinis et al., 1996). We believe that phosphorylation has some role in the IGF-independent effects of IGFBP-3.
In an attempt to further understand the mechanisms that allow for these opposing effects of IGFBP-3 we have investigated the interaction of IGFBP-3 with breast cancer cell membranes. In addition to proteolysis we have recently reported that IGFBP-3 is phosphorylated by breast cancer cells by a process that occurs on the cell membranes, does not require internalization and is inhibited by IGF-I (Mishra and Murphy, 2003, Endocrinology 144:4042–4050). Phosphorylation of IGFBP-3 by this membrane-associated kinase enhanced the binding affinity of IGFBP-3 for IGF-I (Mishra and Murphy, 2003, Endocrinology 144:4042–4050). Thus phosphorylation of IGFBP-3 at the membrane favors the interaction of IGF-I with IGFBP-3 rather than the IGF-I receptor. Furthermore, since formation of IGF-I/IGFBP-3 complexes inhibits binding of IGFBP-3 to the cell membrane, phosphorylation of IGFBP-3 may modulate its pro-apoptotic anti-proliferative effects. To further understand the role of this kinase in physiological regulation of IGFBP-3 action we purified this kinase activity from T47D breast cancer cells. We subsequently demonstrated that this kinase activity is attributed to TG2.
Tissue transglutaminase (TG2) is a ubiquitous enzyme that is involved in post-translation modification and protein-protein interactions. It functions to cross-link glutamine residues with lysine residues resulting in protein polymerization, cross-linking of dissimilar proteins, and incorporation of diamines and polyamines into proteins. It has not previously been known to have kinase activity. In our recent report (Mishra and Murphy, 2003, Endocrinology 144:4042–4050) we demonstrated that insulin-like growth factor binding protein-3 (IGFBP-3) was phosphorylated by breast cancer cell membranes and that this activity was due to TG2. Antiserum to TG2 and protein A-sepharose were used to immunoprecipitate TG2 from IGFBP-3 affinity purified membrane fractions. The immunoprecipitates retained IGFBP-3 kinase activity whereas immunoprecipitation deleted kinase activity in the membrane supernatant. The inhibitors of TG2, cystamine and monodansyl cadaverine, abolished the ability of the T47D cell membrane preparation to phosphorylate IGFBP-3. Both TG2 purified from guinea pig liver and recombinant human TG2 expressed in insect cells were able to phosphorylate IGFBP-3 in vitro. TG2 kinase activity was inhibited in a concentration dependent fashion by calcium, which has previously been shown to be important for the cross-linking activity of TG2. These data provide compelling evidence that TG2 has intrinsic kinase activity, a function that has not previously been ascribed to TG2. Furthermore we provide evidence that TG2 is a major component of the IGFBP-3 kinase activity present on breast cancer cell membranes.
Although TG2 contains a GTP binding domain and can hydrolyse both GTP and ATP (Lai et al., 1998, J. Biol. Chem., 273:1776–1781) it has not previously been reported to have kinase activity. It has however been reported to be involved in apoptosis (Thomazy & Davies, 1999 Cell Death Differ., 6:146–154).
TG2 belongs to a family of nine evolutionary related genes that catalyze the posttranscriptional modification of proteins by inserting an isopeptide bond within or between polypeptide chains. None of the these transglutaminase family members have previously reported to have kinase activity. Although we initially demonstarted that the most abundant member of this family, namely TG2 has intrinsic kinase activity directed towards IGFBP-3 we have also shown that another member of the TG family, namely human coagulation factor XIIIa has kinase activity directed against IGFBP-3. This makes it a reasonable prediction that all members of the TG family have this kinase activity. Furthermore we have shown that the kinase activity of TG2 is not restricted to IGFBP-3 but other substrates as well. TG2 also phosphorylated the tumor suppressor genes p53 and retinoblastoma protein (pRb) and histone H3 (FIG. 5). These proteins have been shown to be critically important in cellular proliferation and disturbances in their expression and/or function is apparent in many cancers and disease states associated with increased cell proliferation.
Although the cross-linking activites of TG2 have been thought important in apoptosis this has not been definitively demonstrated. We have shown that the calcium, which stimulates the cross-linking activites of TG2 actually inhibits the kinase activity and therefore we propose that calcium acts as a switch to change the function of TG2 from a kinase to a cross-linking enzyme.